Mannan-binding lectin (mbl) treatment of infections in individuals treated with tnf-alphainhibitors

ABSTRACT

The present invention relates to the use of subunits and oligomers of mannan-binding lectin (MBL) in prophylactic and/or curative treatment of infections in an individual receiving TNF-inhibiting treatment, in particular TNF-α-inhibiting treatment. The MBL may be administered together with an antimicrobial agent, for example as a kit-of-parts.

[0001] The present invention pertains to the use of subunits andoligomers of mannan-binding lectin (MBL) in prophylactic and/or curativetreatment of infections in an individual receiving TNF-inhibitingtreatment.

[0002] Tumor Necrosis Factor: Monocytes and macrophages secretecytokines known as tumor necrosis factor-α (TNF-α) and tumor necrosisfactor-.beta. (TNF.beta.) in response to endotoxin or other stimuli.TNF-α is a soluble homotrimer of 17 kD protein subunits (Smith, et al.,J. Biol. Chem. 262:6951-6954 (1987)). A membrane-bound 26 kD precursorform of TNF also exists (Kriegler, et al., Cell 53:45-53 (1988)). Forreviews of TNF, see Beutler, et al., Nature 320:584 (1986), Old, Science230:630 (1986), and Le, et al., Lab. Invest. 56:234 (1987).

[0003] Cells other than monocytes or macrophages also make TNF-α. Forexample, human non-monocytic tumor cell lines produce TNF (Rubin, etal., J. Exp. Med. 164:1350 (1986); Spriggs, et al., Proc. Natl. Acad.Sci. USA 84:6563 (1987)). CD4.sup.+ and CD8.sup.+ peripheral blood Tlymphocytes and some cultured T and B cell lines (Cuturi, et al., J.Exp. Med. 165:1581 (1987); Sung, et al., J. Exp. Med. 168:1539 (1988))also produce TNF-α.

[0004] TNF causes pro-inflammatory actions which result in tissueinjury, such as inducing procoagulant activity on vascular endothelialcells (Pober, et al., J. Immunol. 136:1680 (1986)), increasing theadherence of neutrophils and lymphocytes (Pober, et al., J. Immunol.138:3319 (1987)), and stimulating the release of platelet activatingfactor from macrophages, neutrophils and vascular endothelial cells(Camussi, et al., J. Exp. Med. 166:1390 (1987)).

[0005] Recent evidence associates TNF with infections (Cerami, et al.,Immunol. Today 9:28 (1988)), immune disorders, neoplastic pathologies(Oliff, et al., Cell 50:555 (1987)), autoimmune pathologies andgraft-versus host pathologies (Piguet, et al., J. Exp. Med. 166:1280(1987)). The association of TNF with cancer and infectious pathologiesis often related to the host's catabolic state. Cancer patients sufferfrom weight loss, usually associated with anorexia.

[0006] The extensive wasting which is associated with cancer, and otherdiseases, is known as “cachexia” (Kern, et al. (J. Parent. Enter. Nutr.12:286-298 (1988)). Cachexia includes progressive weight loss, anorexia,and persistent erosion of body mass in response to a malignant growth.The fundamental physiological derangement can relate to a decline infood intake relative to energy expenditure. The cachectic state causesmost cancer morbidity and mortality. TNF can mediate cachexia in cancer,infectious pathology, and other catabolic states.

[0007] TNF also plays a central role in gram-negative sepsis andendotoxic shock (Michie, et al., Br. J. Surg. 76:670-671 (1989); Debets,et al., Second Vienna Shock Forum, p.463-466 (1989); Simpson, et al.,Crit. Care Clin. 5:27-47 (1989)), including fever, malaise, anorexia,and cachexia. Endotoxin strongly activates monocyte/macrophageproduction and secretion of TNF and other cytokines (Kombluth, et al.,J. Immunol. 137:2585-2591 (1986)). TNF and other monocyte-derivedcytokines mediate the metabolic and neurohormonal responses to endotoxin(Michie, et al., New. Engl. J. Med. 318:1481-1486 (1988)). Endotoxinadministration to human volunteers produces acute illness with flu-likesymptoms including fever, tachycardia, increased metabolic rate andstress hormone release (Revhaug, et al., Arch. Surg. 123:162-170(1988)). Circulating TNF increases in patients suffering fromGram-negative sepsis (Waage, et al., Lancet 1:355-357 (1987); Hammerle,et al., Second Vienna Shock Forum p. 715-718 (1989); Debets, et al.,Crit. Care Med. 17:489-497 (1989); Calandra, et al., J. Infect. Dis.161:982-987 (1990)).

[0008] The numerous biological effects of TNF-α and the closely relatedcytokine, TNF.beta. (lymphotoxin), are mediated by two TNF transmembranereceptors, both of which have been cloned. The p55 receptor (also termedTNF-R55, TNF-RI, or TNFR.beta.) is a 55 kd glycoprotein shown totransduce signals resulting in cytotoxic, anti-viral, and proliferativeactivities of TNF-α.

[0009] The p75 receptor (also termed TNF-R75, TNF-RII, or TNFRα) is a 75kDa glycoprotein that has also been shown to transduce cytotoxic andproliferative signals as well as signals resulting in the secretion ofGM-CSF. The extracellular domains of the two receptors have 28% homologyand have in common a set of four subdomains defined by numerousconserved cysteine residues. The p75 receptor differs, however, byhaving a region adjacent to the transmembrane domain that is rich inproline residues and contains sites for O-linked glycosylation.Interestingly, the cytoplasmic domains of the two receptors share noapparent homology which is consistent with observations that they cantransduce different signals to the interior of the cell.

[0010] TNF-α inhibiting proteins have been detected in normal humanurine and in serum of patients with cancer or endotoxemia. These havesince been shown to be the extracellular domains of TNF receptorsderived by proteolytic cleavage of the transmembrane forms. Many of thesame stimuli that result in TNF-α release also result in the release ofthe soluble receptors, suggesting that these soluble TNF-α inhibitorscan serve as part of a negative feedback mechanism to control TNF-αactivity.

[0011] Aderka, et al., Isrl. J. Med. Sci. 28:126-130 (1992) disclosessoluble forms of TNF receptors (sTNF-Rs) which specifically bind TNF andthus can compete with cell surface TNF receptors to bind TNF (Seckinger,et al., J. Exp. Med. 167:1511-1516 (1988); Engelmann, et al., J. Biol.Chem. 264:11974-11980 (1989)).

[0012] Anti-TNFα treatment has been suggested to treat a variety ofpatients suffering from an inflammatory disease, such as Crohn'sdisease, ulcerative colitis, arthritis, rheumatoid arthritis. Vide forexample U.S. Pat. No. 5,919,542. TNF-alpha inhibitor therapy resultsamong other things in reduced recruitment of of neutrophils to the siteof infection (reduced secretion of GM-CSF) and reduced functionality ofneutrophils (inhibition of neutrophils adherance). Further, it has beenreported that some of the patients treated with anti-TNFα treatment havedeveloped severe infections, some even fatal infections.

SUMMARY OF THE INVENTION

[0013] A subpopulation of patients receiving TNF-α inhibitor treatmentof inflammatory diseases as discussed above suffer from severeinfections after onset of the TNF-α inhibitor treatment.

[0014] By the present invention MBL treatment and/or prophylaxis of theTNF-α inhibitor treatment induced infections is suggested.

[0015] Mannan-binding lectin (MBL), synonymous to mannose-bindinglectin, mannan-binding protein or mannose-binding protein (MBP), belongsto a subgroup of C-type lectins, termed collecting, since these solubleproteins are composed of subunits presenting three CRDs attached to acollagenous stalk². MBL interact with carbohydrates presented by a widerange of micro-organisms and accumulating evidence shows that it playsan important role in the innate immune defence³. When bound tocarbohydrate MBL is able to activate the complement system.

[0016] The complement system may be activated via three differentpathways: the classical pathway, the alternative pathway, and the newlydescribed third pathway, the mannan-binding lectin (MBL) pathway whichis initiated by the binding of MBL to carbohydrates presented bymicro-organisms. The components of the alternative pathway and of theMBL pathway are parts of the innate immune defence, also termed thenatural or the non-clonal, immune defence, while the classical pathwayinvolves cooperation with antibodies of the specific immune defence.

[0017] The human MBL protein is composed of up to 18 identical 32 kDapolypeptide chains²⁷, each comprising a short N-terminal segment of 21amino acids including three cysteine residues, followed by 7 repeats ofthe collagenous motif Gly-X-Y interrupted by a Gln residues followed byanother 12 Gly-X-Y repeats. A small 34 residue ‘neck-region’ joins theC-terminal Ca²⁺-dependent lectin domain of 93 amino acids with thecollagenous part of the molecule²⁸.

[0018] The collagenous regions of the three polypeptide chains combineto form a subunit which is stabilised covalently by disulphide bridges.Individual subunits are joined by disulphide bridges as well as bynon-covalently interactions²⁷.

[0019] The concentration of MBL in human serum is largely geneticallydetermined, but reportedly increases up to threefold during acute phasereactions⁸. Three mutations causing structural alterations and twomutations in the promotor region are associated with MBL deficiency⁹.MBL deficiency is associated with susceptibility to a variety ofinfections.

[0020] A wide range of oligosaccharides can bind to MBL. As the targetsugars are not normally exposed on mammalian cell surfaces at highdensities, MBL does not usually recognize self-determinants, but isparticularly well suited to interactions with microbial cell surfacespresenting repetitive carbohydrate determinants. In vitro, yeast(Candida albicans and Cryptococcus neoformans), viruses (HIV-1, HIV-2,HSV-2, and various types of influenza A) and a number of bacteria havebeen shown to be recognized by MBL. In the case of some bacteria, thebinding with MBL is impaired by the presence of a capsule¹³. However,even encapsulated bacteria (Neisseria meningitidis) can show strongbinding of MBL¹⁴.

[0021] Thus, the invention features the use of MBL, purified fromnatural sources or from material produced by recombinant technologies,or by any other suitable MBL-producing cell line, for the prophylaxisand/or treatment of infections associated with a therapeutical ormedical treatment with TNF-α inhibitors. The MBL may be given before orafter start of the medical treatment and for any duration of time deemedsuitable.

[0022] By the term TNF-α inhibitors is meant any treatment havingneutralizing and/or inhibiting activity against TNF-α. A TNF-α inhibitormay be a compound or a composition comprising anti-TNF-α antibodies,and/or anti-TNF-α peptides. Typically the TNF-α inhibitors bind to TNF-αblocking its interaction with cell surface TNF receptors therebyneutralizing and/or inhibiting the activity of TNF-α.

[0023] Also TNF-α inhibitors may be peptides or often compounds capableof binding to the TNF-α thereby inhibiting and/or neutralizing thebiological activity of TNF-α. Examples of peptides are soluble TNF-αreceptor complex or fragments thereof. Also, combinations of antibodieswith TNF-α receptors or fragments thereof have been used as TNF-αinhibitors.

[0024] For example a chimeric monoclonal antibody comprised of humanconstant and murine variable regions, an example hereof is a drug“REMICADE” from Centocor, Inc., binding specifically to TNF-α.

[0025] Another example of a TNF-α inhibitor is dimeric fusion proteinconsisting of the extracellular ligand-biding portion of the human 75kilodalton tumor necrosis factor receptor linked to the Fc portion ofhuman IgG1. An example hereof is the drug “ENBREL” fromImmunexCorporation, wherein the Fc component of etnercept contains theC_(H) 2 domain, the C_(H) 3 domain and the hinge region, but not theC_(H) 1 domain of IgG1.

[0026] Another example of TNF-α inhibitor therapy is the use oftolerance breaking vaccines, as those developed by M&E Biotech A/S(since May 16, 2001 Pharmexa), capable of inducing autoantibodiesdirected against TNF-α.

[0027] MBL is believed to exert its antimicrobial activity mainlythrough its opsonizing activity (preparation of microorganisms forphagocytosis). This activity is dependent on activation of complementafter binding of MBL to the microbial surface and deposition of C4b andC3b on the microorganism. MBL can also promote the directcomplement-mediated killing of the microorganism through the activationof the terminal lytic pathway of complement and insertion of themembrane attack complex (MAC) in the membrane. This mechanism isconsidered of minor importance. Many microorganisms, such asGram-positive bacteria, e.g., Streptococcus pneumonia, are resistant toMAC, but can be eliminated by opsonophagocytosis. Consideringopsonophagocytosis as the main effector mechanism of MBL-mediatedclearance of microorganisms, it is a surprise that MBL treatment couldbe of benefit to persons having an impaired phagocytic function due toTNF-α inhibitor therapy, in particular an impaired phagocytic functionat a site of infection due to TNF-α inhibitor therapy.

[0028] It is possible according to the invention to prophylacticallytreat an infection in an individual receiving TNF-α inhibitor treatment.By prophylactically treating with MBL before or during the treatment itis possible to prevent a subsequent infection or to reduce the risk ofthe individual contracting an infection.

[0029] The invention is also directed to treatments of such deficienciesby infusion of MBL. Furthermore, the invention is directed to the use ofMBL plasma concentrations for predicting the risk of infection ofindividuals undergoing therapy with TNF-α inhibitors.

[0030] In another aspect the present invention is related to the use ofa composition comprising at least one mannan-binding lectin (MBL)subunit, or at least one oligomer comprising the at least onemannan-binding lectin (MBL) subunit, in the manufacture of a medicamentfor prophylactic, ameliorating or curative treatment of an infection inan individual initially having low plasma levels of MBL, such as plasmalevels of about 0 mg/ml, or plasma levels in excess of 10 ng/. Inparticular the individual may be genetically disposed to an MBLdeficiency or have acquired an MBL deficiency leading to an increasedrisk of suffering from infections. Accordingly, the invention alsoconcerns treatment of infections in individuals suffering from amannan-binding lectin (MBL) deficiency including any deficiency in theproduction of MBL and/or function of MBL, in particular however,individuals which furthermore receive treatment with a TNF-α inhibitor.

[0031] In yet another aspect there is provided a method for estimatingthe probability of the occurrence of any clinically significantinfection in an individual undergoing therapy with TNF-α inhibitors,said method comprising the step of measuring the concentration of MBL inplasma or serum obtained from the individual, and estimating theprobability on the basis of the measured concentration.

[0032] Also, by genotyping the individuals in question it is possible toestimate the probability of the occurrence of any clinically significantinfection in an individual undergoing therapy with TNF-α inhibitors.

DETAILED DESCRIPTION OF THE INVENTION

[0033] TNF related pathologies include, but are not limited to, thefollowing:

[0034] (A) acute and chronic immune and autoimmune pathologies, such assystemic lupus erythematosus (SLE) rheumatoid arthritis, thyroidosis,graft versus host disease, scleroderma, diabetes mellitus, Graves'disease, and the like;

[0035] (B) infections, including, but not limited to, sepsis syndrome,cachexia, circulatory collapse and shock resulting from acute or chronicbacterial infection, acute and chronic parasitic and/or infectiousdiseases, bacterial, viral or fungal, such as a HIV, AIDS (includingsymptoms of cachexia, autoimmune disorders, AIDS dementia complex andinfections);

[0036] (C) inflammatory diseases, such as chronic inflammatorypathologies and vascular inflammatory pathologies, including chronicinflammatory pathologies such as sarcoidosis, chronic inflammatory boweldisease, ulcerative colitis, and Crohn's pathology and vascularinflammatory pathologies, such as, but not limited to, disseminatedintravascular coagulation, atherosclerosis, and Kawasaki's pathology:

[0037] (D) neurodegenerative diseases, including, but are not limitedto,

[0038] demyelinating diseases, such as multiple sclerosis and acutetransverse myelitis;

[0039] extrapyramidal and cerebellar disorders' such, as lesions of thecorticospinal system;

[0040] disorders of the basal ganglia or cerebellar disorders;

[0041] hyperkinetic movement disorders such as Huntington's Chorea andsenile chorea;

[0042] drug-induced movement disorders, such as those induced by drugswhich block CNS dopamine receptors;

[0043] hypokinetic movement disorders, such as Parkinson's disease;

[0044] Progressive supranucleo palsy;

[0045] Cerebellar and Spinocerebellar Disorders, such as astructurallesions of the cerebellum;

[0046] spinocerebellar degenerations (spinal ataxia, Friedreich'sataxia, cerebellar cortical degenerations, multiple systemsdegenerations (Mencel, Dejerine-Thomas, Shi-Drager, and Machado-Joseph);and systemic disorders (Refsum's disease, abeta-lipoprotemia, ataxia,telangiectasia, and mitochondrial multi.system disorder);

[0047] demyelinating core disorders, such as multiple sclerosis, acutetransverse myelitis; disorders of the motor unit, such as neurogenicmuscular atrophies (anterior horn cell degeneration, such as amyotrophiclateral sclerosis, infantile spinal muscular atrophy and juvenile spinalmuscular atrophy); Alzheimer's disease; Down's Syndrome in middle age;Diffuse Lewy body disease; Senile Dementia of Lewy body type;Wernicke-Korsakoff syndrome; chronic alcoholism; Creutzfeldt-Jakobdisease; Subacute sclerosing panencephalitis, Hallerrorden-Spatzdisease; and Dementia pugilistica, or any subset thereof;

[0048] (E) malignant pathologies involving TNF-secreting tumors or othermalignancies involving TNF, such as, but not limited to leukemias(acute, chronic myelocytic, chronic lymphocytic and/or myelodyspasticsyndrome); lymphomas (Hodgkin's and non-Hodgkin's lymphomas, such asmalignant lymphomas (Burkitt's lymphoma or Mycosis fungoides)); and

[0049] (F) alcohol-induced hepatitis.

[0050] See, e.g., Berkow et al, eds., The Merck Manual, 16th edition,chapter 11, pp 1380-1529, Merck and Co., Rahway, N.J., 1992, whichreference, and references cited therein, are entirely incorporatedherein by reference.

[0051] Such treatment comprises parenterally administering a single ormultiple doses of the antibody, fragment or derivative. Preferred forhuman pharmaceutical use are high affinity potent hTNF-α-inhibitingand/or neutralizing murine and chimeric antibodies, fragments andregions.

[0052] Anti-TNF peptides or MAbs can be administered by any means thatenables the active agent to reach the agent's site of action in the bodyof a mammal. In the case of the antibodies of this invention, theprimary focus is the ability to reach and bind with TNF released bymonocytes and macrophages or other TNF producing cells. Because proteinsare subject to being digested when administered orally, parenteraladministration, i.e., intravenous, subcutaneous, intramuscular, wouldordinarily be used to optimize absorption.

[0053] Infections related to TNF-α inhibitor treatment may be preventedand/or treated in individuals independent on their serum MBL level. Inparticular infections may be prevented when administering MBL to theseindividuals having an MBL level in excess of 10 ng/ml serum. Also,individuals having an MBL level in excess of 50 ng/ml serum may be inneed of treatment, such as individuals having an MBL level in excess of100 ng/ml serum, and individuals having an MBL level in excess of 150ng/ml serum.

[0054] Also the MBL treatment of infections may be conducted byadministering MBL to these individuals in combination with relevantantibiotics, anti-viral agents or anti-fungal agents.

[0055] In particular, individuals at risk of acquiring infectionresulting from treatment with TNF-α inhibitors will benefit from beingprophylactically treated with MBL before, during and maybe also afterthe treatment in order to prevent infections associated with orresulting from the anti TNF-α treatment.

[0056] Generally all individuals being treated with TNF-α inhibitorsshould be treated with MBL independent on their specific MBL level. Thereason behind this is that infection may lead to MBL depletion, andtherefore an MBL “booster”, increasing the MBL level initially willreduce the risk of MBL depletion to a level below a deficiency level,and the immune defence of these patients can be reinforced byadministration of recombinant or natural plasma-derived MBL. Inparticular infections may be prevented when administering MBL toindividuals having an MBL level in excess of 10 ng/ml serum. Also,individuals having an MBL level in excess of 50 ng/ml serum may be inneed of treatment, such as individuals having an MBL level in excess of100 ng/ml serum, and individuals having an MBL level in excess of 150ng/ml serum.

[0057] The present inventors have also shown herein that in particularindividuals having an MBL level below 500 ng/ml serum will benefit fromthe MBL treatment. Consequently, in particular individuals having an MBLlevel below 400 ng/ml will benefit, such as individuals having an MBLlevel below 300 ng/ml, such as individuals having an MBL level below 250ng/ml, such as individuals having an MBL level below 200 ng/ml.

[0058] Thus, in a preferred embodiment the present invention relates tothe use of MBL for manufacturing of a medicament for treatment ofindividuals having an MBL level in serum in the range of 10-500 ng/ml,such as in the range of 50-500 ng/ml for treating and/or preventinginfections.

[0059] One group of individuals being in need of MBL treatment in orderto prevent and/or treat infections are individuals having a low level offunctional MBL, independent on the level of MBL as such. This is due tothe fact, that for some mutations of the MBL it has been found, thatalthough MBL subunits and oligomers thereof are expressed in serum thefunctionality thereof are low. The functionality or functional activityof MBL may be estimated by its capacity to form an MBL/MASP complexleading to activation of the complement system. When C4 is cleaved byMBL/MASP an active thiol-ester is exposed and C4 becomes covalentlyattached to nearby nucleophilic groups. A substantial part of the C4bwill thus become attached to the coated plastic well and may be detectedby anti-C4 antibody.

[0060] A quantitative TRIFMA for MBL functional activity is constructedby 1) coating microtitre wells with 1 mg mannan in 100 ml buffer; 2)blocking with Tween-20; 3) applying test samples, e.g. diluted MBLpreparations 4) applying MBL deficient serum (this leads to theformation of the MBL/MASP complex); alternatively the MBL and the MBLdeficient serum may be mixed before application with the microtitrewells; 5) applying purified complement factor C4 at 5 mg/ml; 6) incubatefor one hour at 37° C.; 7) applying Eu-labelled anti-C4 antibody; 8)applying enhancement solution; and 9) reading the Eu by time resolvedfluorometry. Between each step the plate is incubated at roomtemperature and washed, except between step 8 and 9.

[0061] Estimation by ELISA may be carried out similarly, e.g. byapplying biotin-labelled anti-C4 in step 7; 8) apply alkalinephosphatase-labelled avidin; 9) apply substrate; and 10) read the colourintensity.

[0062] The functionality may be expressed as the specific activity ofMBL, such as 1 unit of MBL activity per ng MBL. A non-functional MBL maybe defined as MBL having a specific activity less than 50% of plasma MBLspecific activity, such as less than 25% of plasma MBL specificactivity, wherein the plasma MBL is purified from an individual notsuffering from any MBL mutations. In particular the reference plasma MBLis plasma pool LJ 6.57 Apr. 28, 1997.

[0063] Thus, the present invention also relates to the prevention and/ortreatment of infection in individuals having a mutation in their MBLgene leading to a reduced expression of MBL and/or expression ofnon-functional MBL.

[0064] In particular such mutations in the MBL gene can lead to a changeof aminoacid number 52 (numbering including the leader peptide of MBL)from arginine to cysteine, aminoacid number 54 from glycine to asparticacid or amino acid number 75 from glycine to glutamic acid.

[0065] Also mutations in the promoter region of the MBL gene can lead tolowered levels of MBL. In particular mutations at position −221 have aninfluence on the expression of MBL.

[0066] The MBL sequence may be found in swiss.prot under accession No:11226

[0067] The MBL composition used to manufacture an MBL medicament may beproduced from any MBL source available. The MBL source may be naturalMBL, whereby the MBLs are produced in a native host organism, meaningthat MBL is produced by a cell normally expressing MBL. One usual methodof producing an MBL composition is by extraction of MBL from human bodyliquids, such as serum or plasma, but MBL may also be harvested fromcultures of hepatocytes.

[0068] In another aspect the MBL oligomers are produced by a hostorganism not natively expressing an MBL polypeptide, such as byrecombinant technology.

[0069] In a first embodiment the MBL source may be serum, from which anMBL composition is obtained by purification from serum, plasma, milkproduct, colostrum or the like by a suitable purification method, suchas affinity chromatography using carbohydrate-derivatised matrices, suchas mannose or mannan coupled matrices. Such a method is discussed inWO99/64453, wherein the purification process is followed by avirus-removal step in order to remove infectious agents from the MBLsource, since one of the major problems with proteins purified from bodyliquids is the risk of introducing infectious agents in combination withthe desired protein. WO99/64453 is hereby incorporated by reference.

[0070] The MBL composition used to manufacture an MBL medicamentpreferably comprises MBL oligomers having a size distributionsubstantially identical to the size distribution of MBL in serum, suchas a size distribution profile at least 50% identical to the sizedistribution profile of MBL in serum. By identical is meant that atleast 50% of the oligomers has an apparent molecular weight higher than200 kDa, when analysed by SDS-PAGE and/or Western blot.

[0071] In a more preferred embodiment the size distribution profile isat least 75% identical to the size distribution profile of MBL in serum,such as at least 90% identical to the size distribution profile of MBLin serum, and more preferred at least 95% identical to the sizedistribution profile of MBL in serum.

[0072] When purifying from an MBL source initially having another sizedistribution profile it is preferred that the affinity chromatographyused to purify from the MBL source favours purification of oligomershaving an apparent molecular weight higher than 200 kDa. This isobtained by using a carbohydrate-derivatized matrix having substantiallyno affinity to subunits and/or dimers of MBL. Preferably thecarbohydrate-derivatized matrix has affinity for substantially onlytetrameric, pentameric and/or hexameric recombinant MBLs.

[0073] The matrix may be derivatized with any carbohydrate orcarbohydrate mixture whereto MBL binds and for which binding of thehigher oligomers of MBL are favoured. The carbohydrate-derivatizedmatrix is preferably a hexose-derivatized matrix, such as a mannose- ora N-acetyl-glucosamin derivatized matrix, such as most preferably amannose-derivatized matrix.

[0074] The selectivity of the carbohydrate-derivatized matrix isobtained by securing that the matrix as such, i.e the un-derivatizedmatrix has substantially no affinity to MBL polypeptides, in particularno affinity to MBL trimers or smaller oligomers. This may be ensuredwhen the matrix as such is carbohydrate-free. In particular the matrixshould not contain any Sepharose or the like. It is preferred that thematrix consists of a non-carbohydrate containing polymer material, suchas Fractogel®TSK beads

[0075] The matrix may be in any form suitable for the chromatography,mostly in the form of beads, such as plastic beads.

[0076] After application of the MBL source the column is washed,preferably by using non-denaturing buffers, having a composition, pH andionic strength resulting in elimination of proteins, without eluting thehigher oligomers of MBL. Such as buffer may be TBS. Elution of MBL isperformed with a selective desorbing agent, capable of efficient elutionof highed oligomers of MBL, such as TBS comprising a desorbing agent,such as EDTA (for example 5 mM EDTA) or mannose (for example 50 mMmannose), and MBL oligomers are collected. Such a purification method isdescribed in co-pending International patent application having thetitle “Recombinant Human Mannan Binding Lectin” filed the same day asthe present application.

[0077] In a preferred aspect a clinical grade MBL composition isobtained by using an MBL source produced by recombinant technology,wherein the MBL source is the culture media from culturing of MBLproducing cells.

[0078] Thus, the present invention encompasses MBL produced by a processof producing a recombinant mannan binding lectin (MBL), comprising thesteps of:

[0079] preparing a gene expression construct comprising a DNA sequenceencoding a MBL polypeptide or a functional equivalent thereof,

[0080] transforming a host cell culture with the construct,

[0081] cultivating the host cell culture, thereby obtaining expressionand secretion of the polypeptide into the culture medium, followed by

[0082] obtaining a culture medium comprising human recombinant MBLs.

[0083] The culture medium comprising the human recombinant MBLpolypeptides may then be processed as described above for purificationof MBL.

[0084] The MBL polypeptide is preferably a mammalian MBL polypeptide,such as more preferably a human MBL polypeptide. The gene expressionconstruct may be produced by conventional methods known to the skilledperson, such as described in U.S. Pat. No. 5,270,199.

[0085] In another embodiment the gene expression construct is preparedas described in PCT/DK00/00246.

[0086] The expression is preferably carried out in e.g. mammalian cells,the preparation according to the invention results from the use of anexpression vector comprising intron sequence(s) from an MBL gene and atleast one exon sequence. Regarding the transgenic animals as expressionsystem this term is in this context animals which have been geneticallymodified to contain and express the human MBL gene or fragments ormimics hereof.

[0087] In addition to the purification method it is preferred that thegene expression construct and the host cell also favours production ofhigher oligomers, which has been found to be possible by using a geneexpression construct comprising at least one intron sequence from thehuman MBL gene or a functional equivalent thereof. malian cells andcells from insects.

[0088] In particular the MBL composition is used for treatment and/orprophylaxis of an infection associated with TNF-α inhibitor treatment inan individual. Any microbiological infections may be treated and/orprevented with MBL, i.e. any infection caused by a microbial species.

[0089] Consequently, the MBL composition may be used for preventingand/or treating an infection in an individual wherein the microbialspecies is a fungus, a yeast, a protozoa, a parasite and/or a bacteria.

[0090] Also, the MBL composition may be used for treating infection,wherein the microbial species is resistent to usual medicaments, such asinfections for which the bacterial species is resistent to at least oneantibiotic medicament. More important is the prophylaxis and/ortreatment of infections for which the bacterial species ismultiresistent.

[0091] The individuals may suffer from infections caused by pathogenicbacterial species, such as Streptococcus pneumonia, Salmonella andStaphylococcal species.

[0092] It is however well-known that in particular immuno-compromisedindividuals also often suffer from infections caused by bacterialspecies, that are normally nonpathogenic, i.e. opportunistic pathogens,e.g. E. coli species, and many of these species are resistent to usualantibiotic treatment.

[0093] The infection associated with the condition may also be a viralinfection, such as a viral infection wherein the virus is a retrovirus.

[0094] Also, the immuno-compromised condition may be an infection withthe retrovirus Human Immunodeficiency Virus (HIV). However, the viralinfections treated and/or prevented according to the invention arenormally not caused by a retrovirus, but may for example be caused by aDNA virus.

[0095] Parasites according to the present invention may for example beselected from the group consisting of Malaria (Plasmodium. falciparum,P. vivax, P. malariae), Schistosomes, Trypanosomes, Leishmania, Filarialnematodes, Trichomoniasis, Sarcosporidiasis, Taenia (T. saginata, T.solium), Leishmania, Toxoplasma gondii, Trichinelosis (Trichinellaspiralis) or Coccidiosis (Eimeria species).

[0096] The medicament may be produced by using the eluant obtained fromthe affinity chromatography as such. It is however preferred that theeluant is subjected to further purification steps before being used.

[0097] In addition to the MBL oligomers, the medicament may comprise apharmaceutically acceptable carrier substance and/or vehicles. Inparticular, a stabilising agent may be added to stabilise the MBLproteins. The stabilising agent may be a sugar alcohol, saccharides,proteins and/or aminoacids. Examples of stabilising agents may bemaltose or albumin.

[0098] Other conventional additives may be added to the medicamentdepending on administration form for example. In one embodiment themedicament is in a form suitable for injections. Conventional carriersubstances, such as isotonic saline, may be used.

[0099] In another embodiment the medicament is in a form suitable forpulmonal administration, such as in the form of a powder for inhalationor creme or fluid for topical application.

[0100] The route of administration may be any suitable route, such asintravenously, intramusculary, subcutanously or intradermally. Also,pulmonal or topical administration is envisaged by the presentinvention.

[0101] The MBL composition may also be administered simultaneously,sequentially or separately with the TNF-α inhibitor treatment. Themedicament may be administered for a period before the onset ofadministration of TNF-α-inhibitor and/or during at least a part of theTNF-α-inhibitor treatment.

[0102] The MBL composition is administered in suitable dosage regimes,in particularly it is administered repeatedly at suitable intervals,such as once or twice a week, starting before onset of TNF-α-inhibitortherapy and maintained at intervals, for example once a week, at leastduring a part of the therapy period, preferably during the whole therapyperiod.

[0103] Normally from 1-100 mg is administered per dosage, such as from2-10 mg, mostly from 5-10 mg per dosage depending on the individual tobe treated, for example about 0.1 mg/kg body weight is administered.

[0104] The use of an MBL composition for the manufacture of a medicamentmay also further comprise the manufacture of another medicament, such asan anti-fungal, anti-yeast, anti-bacterial and/or anti-viral medicamentfor obtaining a kit-of.parts.

[0105] The anti-viral medicament may be a medicament capable of virusattenuation and/or elimination.

[0106] The invention also relates to an aspect of using a measurement ofthe MBL level as a prognostic marker for the risk of the individual ofacquiring an infection, and thereby an indicative of the need fortreatment. In particular an MBL level below 500 ng/ml is a prognosticmarker indicative for treatment with MBL, in particular in an individualthat receives and/or will receive and/or have received treatment with aTNF-α inhibitor.

[0107] The prognostic marker may be in relation to any infection, but isespecially relevant as a prognostic marker for septicaemia or pneumoniain individuals undergoing TNF-α inhibitor therapy.

[0108] Thus, the present invention also relates to a method of using anMBL composition for preventing and/or reducing infections in anindividual, the method comprising the steps of:

[0109] i) determining serum levels of MBL in an individual,

[0110] ii) estimating the probability of the occurence of a significantclinical infection in the individual, and optionally,

[0111] administering an MBL composition to the individual.

[0112] The MBL level is measured in serum or plasma, and may bedetermined by time resolved immunofluorescent assay (TRIFMA), ELISA, RIAor nephelometry.

[0113] Also the MBL levels may be inferred from analysis of genotypes ofthe MBL genes as discussed above in relation to mutations of MBL leadingto a decreased MBL level.

EXAMPLE

[0114] MBL Serum Levels in Patients Treated with TNF-α Inhibitors

[0115] Patients are selected among individuals that have receivedtreatment with either etanercept or infliximab. Patients presentingclinically significant infections (CSI, defined as bacteriaemia orpneumonia) are identified by retrospective computer search of thepatient database.

[0116] Before entering treatment blood is drawn into evacuated glasstubes containing EDTA (final concentration about 10 mM). The plasma isaliquoted and kept at −80° C. until assay. Plasma samples are similarlyobtained from healthy blood donors. The patients are free of infectionsat the time of blood sampling.

[0117] The concentration of MBL is determined by a time resolvedimmunofluorescent assay (TRIFMA). Microtitre wells (fluoroNunc, Nunc,Kamstrup, Denmark) are coated with antibody by incubation overnight atroom temperature with 500 ng anti-human MBL antibody (Mab 131-1, StatensSerum Institut, Copenhagen, Denmark) in 100 μl PBS (0.14 M NaCl, 10 mMphosphate, pH 7.4). After wash with Tween-containing buffer (TBS, 0.14 MNaCl, 10 mM Tris/HCl, 7.5 mM NaN₃, pH 7.4 with 0.05% Tween 20) testsamples (plasma 1/20) and calibrator dilutions are added in TBS/Tweenwith extra NaCl to 0.5 M and 10 mM EDTA.

[0118] After overnight incubation at 4° C. and wash, the developingeuropium-labelled antibody (12.5 ng Mab 131-1 labelled with theEu-containing chelate, isothiocyanato-benzoyl-diethylene-triamine-tetraacetic acid, according to the manufacturer, Wallac, Turku, Finland) isadded in TBS/Tween with 25 μM EDTA.

[0119] Following incubation for 2 h and wash, fluorescence enhancementsolution is added (Wallac) and the plates are read on a time resolvedfluorometre (Delfia 1232, Wallac). The calibration curve is made usingdilutions of one plasma, which is kept alliquoted at −80° C.

[0120] Based on the above outlined method the MBL serum level ofpatients with CSI as compared to non-CSI patients is compared.

[0121] Ref rences

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1. Use of a composition comprising at least one mannan-binding lectin(MBL) subunit, or at least one mannan-binding lectin (MBL) oligomercomprising the at least one mannan-binding lectin (MBL) subunit, in themanufacture of a medicament for prophylaxis and/or treatment ofinfection in an individual being treated with a TNF-α inhibitor.
 2. Theuse according to claim 1, wherein the TNF-α inhibitor is an antibodydirected against TNF-α.
 3. The use according to claim 1, wherein theTNF-α inhibitor is a fusion protein comprising portions of TNF-αreceptor.
 4. The use according to claim 1, wherein the TNF-α inhibitoris selected from etanercept and infliximab.
 5. The use according toclaim 1, wherein the TNF-α inhibitor therapy is vaccination with atolerance breaking TNF-α vaccine resulting in the induction ofautoantibodies against TNF-α.
 6. The use of claim 1, wherein thecomposition comprises at least one mannan-binding lectin (MBL) oligomercomprising the at least one mannan-binding lectin (MBL) subunit.
 7. Theuse of claim 6, wherein said oligomer is preferably selected from thegroup of oligomers consisting of tetramers, pentamers and/or hexamers.8. The use of claim 1, wherein the individual, being at risk ofacquiring an infection resulting from a medical treatment, has a serumlevel of MBL in excess of 10 ng/ml serum.
 9. The use of claim 1, whereinthe individual, being at risk of acquiring an infection resulting from amedical treatment, has a serum level of MBL in excess of 50 ng/ml serum.10. The use of any of claims 8 or 9, wherein the serum MBL level is thefunctional serum MBL level.
 11. The use of claim 1, wherein theinfection is an infection caused by a microbial species.
 12. The use ofclaim 11 wherein the microbial species is a fungus.
 13. The use of claim11, wherein the microbial species is a yeast.
 14. The use of claim 11,wherein the microbial species is a bacteria.
 15. The use of claim 14,wherein the bacterial species is resistent to at least one antibioticmedicament.
 16. The use of claim 14, wherein the bacterial species ismultiresistent.
 17. The use of claim 14, wherein the bacterial speciesis pathogenic.
 18. The use of claim 11, wherein the infection is a viralinfection.
 19. The use of claim 18, wherein the virus is a retrovirus.20. The use of claim 19, wherein the retrovirus is a HumanImmunodeficiency Virus.
 21. The use of claim 1, further comprising themanufacture of an antimicrobial medicament capable of attenuation and/orelimination a microbial species for obtaining a kit-of-parts.
 22. Theuse of claim 21, further comprising the manufacture of an antibacterialmedicament capable of bacterial attenuation and/or elimination forobtaining a kit-of-parts.
 23. The use of claim 1, wherein the MBLsubunit or the MBL oligomer is produced in a native host organism. 24.The use of claim 23, wherein the native host organism is a human cellnatively expressing the MBL subunit or the MBL oligomer.
 25. The use ofclaim 1, wherein the MBL subunit or MBL oligomer is produced by a hostorganism not natively expressing an MBL polypeptide.
 26. The use ofclaim 1, wherein the MBL subunit or the MBL oligomer is produced by amethod comprising at least one step of recombinant DNA technology invitro.
 27. The use of any of claims 25 and 26, wherein the production ofthe MBL subunit or the MBL oligomer is controlled by an expressioncontrol sequence not natively associated with MBL polypeptideexpression.
 28. The use of any of claims 23 to 27, wherein the MBLsubunit or the MBL oligomer is isolated from the host organism.
 29. Theuse of claim 28, wherein the MBL subunit or the MBL oligomer is isolatedby a method comprising at least one step involving affinitychromatography.
 30. The use of claim 29, wherein the affinitychromatography step is capable of isolating MBL tetramers, pentamersand/or hexamers from a composition further comprising additional MBLoligomers and/or MBL subunits.
 31. The use of any of claims 25 to 30,wherein the MBL subunit and/or the MBL oligomer is free from anyimpurities naturally associated with the MBL when produced in a nativehost organism.
 32. The use of claim 1, wherein the MBL subunit is amammalian MBL subunit.
 33. The use of claim 32, wherein the mammalianMBL subunit is a human MBL subunit.
 34. The use of claim 1, wherein themedicament is administered to the individual prior to another treatmentresulting in an infection in the individual.
 35. The use of claim 1,wherein the medicament is administered to the individual simultaneously,sequentially or separately with a TNF-α inhibitor treatment.
 36. The useof claim 35, wherein the medicament is administered to the individualprior to, during and/or after said treatment.
 37. The use of any of thepreceding claims, wherein the treatment is a prophylactic treatment. 38.The use of any of claims 1 to 37, wherein the medicament is a booster ofMBL serum levels in an individual having MBL serum levels above apredetermined minimum MBL serum level of 10 ng/ml.
 39. The use of claim38, wherein the an individual has MBL serum levels below a predeterminedmaximum MBL serum level of 500 ng/ml.
 40. The use of claim 1 or 39,wherein the individual has serum levels of MBL in excess of 75 ng/ml.41. The use of claim 1 or 39, wherein the individual has serum levels ofMBL in excess of 100 ng/ml.
 42. The use of claim 1 or 39, wherein theindividual has serum levels of MBL in excess of 150 ng/ml.
 43. The useof claim 1 or 40, wherein the individual has serum levels of MBL below500 ng/ml.
 44. The use of claim 1 or 40, wherein the individual hasserum levels of MBL below 400 ng/ml.
 45. The use of claim 1 or 40,wherein the individual has serum levels of MBL below 300 ng/ml.
 46. Theuse of any of the preceding claims, wherein serum or plasma levels ofMBL in the individual are determined by quantitative analysis.
 47. Theuse of claim 46, wherein the analysis comprises at least one of ELISA,TRIFMA, RIA or nephelometry.
 48. Method of using an MBL composition forpreventing and/or reducing infections in an individual receiving amedical treatment with a TNF-α inhibitor, the method comprising thesteps of: a) determining serum levels of MBL in an individual, b)estimating the probability of the occurrence of a significant clinicalinfection in the individual, and optionally, c) administering an MBLcomposition to the individual.